The mechanisms of glycolytic inhibition in ischemic myocardium were investigated in the isolated, perfused rat heart. Glycolysis was inhibited at the level of glyceraldehyde-3-phosphate dehydrogenase. The major factors that accounted for the glycolytic inhibition in the ischemic heart compared with the anoxic heart appeared to be higher tissue levels of lactate and H+ in the ischemic tissue. Increased extracellular pH inhibited glycolysis in anoxic and hypoxic hearts much more readily than it did in aerobic hearts. However, maintenance of both extracellular and intracellular pH caused only a modest acceleration of glycolysis in ischemic hearts. Accumulation of tissue lactate and inhibition of glycolysis were directly proportional to the reduction in coronary bloow flow in both anoxic and ischemic hearts. At intracellular lactate concentrations between 15 and 20 mM, glycolysis was inhibited under both conditions. Addition of either 10, 20, or 40 mM lactate to the perfusate inhibited glycolysis in aerobic, anoxic, and ischemic hearts. The effect of lactate did not appear to be mediated through changes in intracellular pH. It is concluded that accumulation of lactate represents a major factor in the inhibition of glycolysis that develops in ischemic hearts.